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1.
Elife ; 92020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32011233

RESUMO

Increasing Neisseria gonorrhoeae resistance to ceftriaxone, the last antibiotic recommended for empiric gonorrhea treatment, poses an urgent public health threat. However, the genetic basis of reduced susceptibility to ceftriaxone is not completely understood: while most ceftriaxone resistance in clinical isolates is caused by target site mutations in penA, some isolates lack these mutations. We show that penA-independent ceftriaxone resistance has evolved multiple times through distinct mutations in rpoB and rpoD. We identify five mutations in these genes that each increase resistance to ceftriaxone, including one mutation that arose independently in two lineages, and show that clinical isolates from multiple lineages are a single nucleotide change from ceftriaxone resistance. These RNA polymerase mutations cause large-scale transcriptional changes without altering susceptibility to other antibiotics, reducing growth rate, or deranging cell morphology. These results underscore the unexpected diversity of pathways to resistance and the importance of continued surveillance for novel resistance mutations.

2.
Nat Microbiol ; 5(2): 291-303, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31932712

RESUMO

Bacteria are protected by a polymer of peptidoglycan that serves as an exoskeleton1. In Staphylococcus aureus, the peptidoglycan assembly enzymes relocate during the cell cycle from the periphery, where they are active during growth, to the division site where they build the partition between daughter cells2-4. But how peptidoglycan synthesis is regulated throughout the cell cycle is poorly understood5,6. Here, we used a transposon screen to identify a membrane protein complex that spatially regulates S. aureus peptidoglycan synthesis. This complex consists of an amidase that removes stem peptides from uncrosslinked peptidoglycan and a partner protein that controls its activity. Amidases typically hydrolyse crosslinked peptidoglycan between daughter cells so that they can separate7. However, this amidase controls cell growth. In its absence, peptidoglycan synthesis becomes spatially dysregulated, which causes cells to grow so large that cell division is defective. We show that the cell growth and division defects due to loss of this amidase can be mitigated by attenuating the polymerase activity of the major S. aureus peptidoglycan synthase. Our findings lead to a model wherein the amidase complex regulates the density of peptidoglycan assembly sites to control peptidoglycan synthase activity at a given subcellular location. Removal of stem peptides from peptidoglycan at the cell periphery promotes peptidoglycan synthase relocation to midcell during cell division. This mechanism ensures that cell expansion is properly coordinated with cell division.

3.
J Biol Chem ; 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974163

RESUMO

Bacteria account for a thousand-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are crosslinked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall tailoring enzymes that regulate glycan strand length, degree of crosslinking, and addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

4.
PLoS Pathog ; 15(11): e1007862, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31738809

RESUMO

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains.

5.
Mol Microbiol ; 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31770461

RESUMO

Staphylococcus aureus is an opportunistic pathogen that can cause soft tissue infections but is also a frequent cause of foodborne illnesses. One contributing factor for this food association is its high salt tolerance allowing this organism to survive commonly used food preservation methods. How this resistance is mediated is poorly understood, particularly during long-term exposure. In this study, we used transposon sequencing (TN-seq) to understand how the responses to osmotic stressors differ. Our results revealed distinctly different long-term responses to NaCl, KCl and sucrose stresses. In addition, we identified the DUF2538 domain containing gene SAUSA300_0957 (gene 957) as essential under salt stress. Interestingly, a 957 mutant was less susceptible to oxacillin and showed increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by amino acid substitutions in the transglycosylase domain of the penicillin-binding protein Pbp2, and these changes restored the peptidoglycan crosslinking to WT levels. These results indicate that increased crosslinking of the peptidoglycan polymer can be detrimental and highlight a critical role of the bacterial cell wall for osmotic stress resistance. This study will serve as a starting point for future research on osmotic stress response and help develop better strategies to tackle foodborne staphylococcal infections.

6.
J Am Chem Soc ; 141(33): 12994-12997, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31386359

RESUMO

The bacterial cell wall is composed of peptidoglycan, and its biosynthesis is an established target for antibiotics. Peptidoglycan is assembled from a glycopeptide precursor, Lipid II, that is polymerized by peptidoglycan glycosyltransferases into glycan strands that are subsequently cross-linked to form the mature cell wall. For decades bacteria were thought to contain only one family of enzymes that polymerize Lipid II, but recently, the ubiquitous Shape, Elongation, Division, and Sporulation (SEDS)-family proteins RodA and FtsW were shown to be peptidoglycan polymerases. Because RodA and FtsW are essential in nearly all bacteria, these enzymes are promising targets for new antibiotics. However, almost nothing is known about the mechanisms of these polymerases. Here, we report that SEDS proteins synthesize peptidoglycan by adding new Lipid II monomers to the reducing end of the growing glycan chain. Using substrates that can only react at the reducing end, we also show that the glycosyl donor and acceptor in the polymerization reaction have distinct lipid requirements. These findings provide the first fundamental insights into the mechanism of SEDS-family polymerases and lay the groundwork for future biochemical and structural studies.

7.
Curr Opin Chem Biol ; 53: 44-50, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31466035

RESUMO

The peptidoglycan cell wall is a unique macromolecular structure in bacteria that defines their shape and confers protection from the surrounding environment. Decades of research has focused on understanding the peptidoglycan synthesis pathway and exploiting its essentiality for antibiotic development. Recently, a new class of peptidoglycan polymerases known as the SEDS (shape, elongation, division and sporulation) proteins were identified; these polytopic membrane proteins function together with the better-known penicillin-binding proteins (PBPs) to build the cell wall. In this review, we will highlight recent developments in chemical tools and methods to label the bacterial cell wall and discuss how these developments are leading to a better understanding of peptidoglycan synthases and their cellular roles.

8.
J Am Chem Soc ; 141(33): 12974-12978, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31373491

RESUMO

O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

9.
Neoplasia ; 21(7): 713-720, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31151054

RESUMO

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism.


Assuntos
Carnitina O-Palmitoiltransferase/genética , Quinase 9 Dependente de Ciclina/genética , Neoplasias da Próstata/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinase 9 Dependente de Ciclina/metabolismo , Ácidos Graxos/metabolismo , Humanos , Masculino , Oxirredução , Fosforilação Oxidativa , Fosforilação , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais/genética
10.
Theranostics ; 9(8): 2183-2197, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31149037

RESUMO

O-GlcNAc transferase (OGT) is overexpressed in aggressive prostate cancer. OGT modifies intra-cellular proteins via single sugar conjugation (O-GlcNAcylation) to alter their activity. We recently discovered the first fast-acting OGT inhibitor OSMI-2. Here, we probe the stability and function of the chromatin O-GlcNAc and identify transcription factors that coordinate with OGT to promote proliferation of prostate cancer cells. Methods: Chromatin immunoprecipitation (ChIP) coupled to sequencing (seq), formaldehyde-assisted isolation of regulatory elements, RNA-seq and reverse-phase protein arrays (RPPA) were used to study the importance of OGT for chromatin structure and transcription. Mass spectrometry, western blot, RT-qPCR, cell cycle analysis and viability assays were used to establish the role of OGT for MYC-related processes. Prostate cancer patient data profiled for both mRNA and protein levels were used to validate findings. Results: We show for the first time that OGT inhibition leads to a rapid loss of O-GlcNAc chromatin mark. O-GlcNAc ChIP-seq regions overlap with super-enhancers (SE) and MYC binding sites. OGT inhibition leads to down-regulation of SE-dependent genes. We establish the first O-GlcNAc chromatin consensus motif, which we use as a bait for mass spectrometry. By combining the proteomic data from oligonucleotide enrichment with O-GlcNAc and MYC ChIP-mass spectrometry, we identify host cell factor 1 (HCF-1) as an interaction partner of MYC. Inhibition of OGT disrupts this interaction and compromises MYC's ability to confer androgen-independent proliferation to prostate cancer cells. We show that OGT is required for MYC-mediated stabilization of mitotic proteins, including Cyclin B1, and/or the increased translation of their coding transcripts. This implies that increased expression of mRNA is not always required to achieve increased protein expression and confer aggressive phenotype. Indeed, high expression of Cyclin B1 protein has strong predictive value in prostate cancer patients (p=0.000014) while mRNA does not. Conclusions: OGT promotes SE-dependent gene expression. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. These features render OGT essential for the androgen-independent, MYC-driven proliferation of prostate cancer cells. Androgen-independency is the major mechanism of prostate cancer progression, and our study identifies OGT as an essential mediator in this process.

11.
Mol Cell ; 75(2): 357-371.e7, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31227231

RESUMO

Carbohydrate response element binding protein (ChREBP) is a key transcriptional regulator of de novo lipogenesis (DNL) in response to carbohydrates and in hepatic steatosis. Mechanisms underlying nutrient modulation of ChREBP are under active investigation. Here we identify host cell factor 1 (HCF-1) as a previously unknown ChREBP-interacting protein that is enriched in liver biopsies of nonalcoholic steatohepatitis (NASH) patients. Biochemical and genetic studies show that HCF-1 is O-GlcNAcylated in response to glucose as a prerequisite for its binding to ChREBP and subsequent recruitment of OGT, ChREBP O-GlcNAcylation, and activation. The HCF-1:ChREBP complex resides at lipogenic gene promoters, where HCF-1 regulates H3K4 trimethylation to prime recruitment of the Jumonji C domain-containing histone demethylase PHF2 for epigenetic activation of these promoters. Overall, these findings define HCF-1's interaction with ChREBP as a previously unappreciated mechanism whereby glucose signals are both relayed to ChREBP and transmitted for epigenetic regulation of lipogenic genes.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteínas de Homeodomínio/genética , Fator C1 de Célula Hospedeira/genética , Lipogênese/genética , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Carboidratos/genética , Epigênese Genética , Regulação da Expressão Gênica , Glucose/metabolismo , Hexosaminas/genética , Hexosaminas/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , Regiões Promotoras Genéticas/genética , Mapas de Interação de Proteínas/genética
12.
Curr Opin Struct Biol ; 56: 97-106, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30708324

RESUMO

Dysregulation of nuclear and cytoplasmic O-linked ß-N-acetylglucosamine (O-GlcNAc) cycling is implicated in a range of diseases including diabetes and cancer. This modification maintains cellular homeostasis by regulating several biological processes, such as cell signaling. This highly regulated cycle is governed by two sole essential enzymes, O-GlcNAc transferase and O-GlcNAcase that add O-GlcNAc and remove it from over a thousand substrates, respectively. Until recently, due to lack of structural information, the mechanism of substrate recognition has eluted researchers. Here, we review recent successes in structural characterization of these enzymes and how this information has illuminated key features essential for catalysis and substrate recognition. Additionally, we highlight recent studies which have used this information to expand our understanding of substrate specificity by each enzyme.

13.
Nat Microbiol ; 4(4): 587-594, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30692671

RESUMO

The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria1. For decades, it was thought that only class A penicillin-binding proteins (PBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA-a member of the unrelated SEDS protein family-also acts as a peptidoglycan polymerase2-4. Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly5,6. FtsW was previously proposed to translocate the peptidoglycan precursor lipid II across the cytoplasmic membrane7,8. Here, we report that purified FtsW polymerizes lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP. We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate class B PBP to produce septal peptidoglycan during cell division.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/enzimologia , Streptococcus thermophilus/enzimologia , Proteínas de Bactérias/genética , Divisão Celular , Parede Celular/genética , Parede Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Ligação às Penicilinas/genética , Ligação Proteica , Staphylococcus aureus/citologia , Staphylococcus aureus/genética , Streptococcus thermophilus/citologia , Streptococcus thermophilus/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-30323039

RESUMO

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward the discovery of such next-generation therapies is to identify novel inhibitors of clinically validated targets, like cell wall biogenesis. We have therefore developed a pathway-directed whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli This conserved multiprotein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam (also known as amdinocillin) to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod- cells. A screen of ∼690,000 compounds identified 1,300 compounds that were active against E. coli Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with those from additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.


Assuntos
Antibacterianos/farmacologia , Parede Celular/metabolismo , Escherichia coli/efeitos dos fármacos , Peptidoglicano/metabolismo , Andinocilina/farmacologia , Andinocilina/toxicidade , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Proteínas de Ligação às Penicilinas/metabolismo
15.
Microbiology ; 165(2): 233-245, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30543507

RESUMO

Streptomyces ghanaensis ATCC14672 is remarkable for its production of phosphoglycolipid compounds, moenomycins, which serve as a blueprint for the development of a novel class of antibiotics based on inhibition of peptidoglycan glycosyltransferases. Here we employed mariner transposon (Tn) mutagenesis to find new regulatory genes essential for moenomycin production. We generated a library of 3000 mutants which were screened for altered antibiotic activity. Our focus centred on a single mutant, HIM5, which accumulated lower amounts of moenomycin and was impaired in morphogenesis as compared to the parental strain. HIM5 carried the Tn insertion within gene ssfg_01967 for putative tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase, or MiaB, and led to a reduced level of thiomethylation at position 37 in the anticodon of S. ghanaensis transfer ribonucleic acid (tRNA). It is likely that the mutant phenotype of HIM5 stems from the way in which ssfg_01967::Tn influences translation of the rare leucine codon UUA in several genes for moenomycin production and life cycle progression in S. ghanaensis. This is the first report showing that quantitative changes in tRNA modification status in Streptomyces have physiological consequences.


Assuntos
Antibacterianos/biossíntese , Genes Bacterianos , Oligossacarídeos/biossíntese , RNA de Transferência/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Isopenteniladenosina/análogos & derivados , Isopenteniladenosina/metabolismo , Mutagênese Insercional , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Esporos Bacterianos , Streptomyces/fisiologia , Sulfurtransferases/genética , Sulfurtransferases/metabolismo
16.
Cancer Nurs ; 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31895175

RESUMO

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare but deadly cancer. Although there is an emerging picture of the individual MPM experience, the United States is underrepresented in this literature. With the United States contributing more deaths from MPM than any other country, findings from this study will enhance a global body of literature on the lived experience of this devastating cancer. OBJECTIVE: The aims of this descriptive phenomenological research study were to explore the lived experience of MPM in the United States and identify unmet patient needs. INTERVENTIONS/METHODS: This was a descriptive phenomenology study employing semi-structured individual interviews with persons with MPM. RESULTS: A total of 7 persons with MPM from a large northeastern US medical center participated. Three major themes about the MPM lived experience emerged: (1) uncertainty/worry about the future, (2) value in relationships, and (3) adapting to a new norm. CONCLUSIONS: Findings from this study are consistent with other MPM research, noting a high symptom burden, lifestyle changes, and feelings of uncertainty about the future. However, participants also expressed feelings of hope and optimism. Particularly salient to the MPM experience was the role of communication with the healthcare team as well as other persons with MPM. IMPLICATIONS FOR PRACTICE: Timely, coordinated, and personalized care as well as skilled communication should be the cornerstone of care for persons with MPM. Supportive care strategies that address uncertainty, the high symptom burden, feelings of isolation, and existential concerns are also integral to quality care.

17.
J Am Chem Soc ; 140(42): 13542-13545, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30285435

RESUMO

Reversible glycosylation of nuclear and cytoplasmic proteins is an important regulatory mechanism across metazoans. One enzyme, O-linked N-acetylglucosamine transferase (OGT), is responsible for all nucleocytoplasmic glycosylation and there is a well-known need for potent, cell-permeable inhibitors to interrogate OGT function. Here we report the structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity. In addition to disclosing useful OGT inhibitors, the structures we report provide insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.


Assuntos
Desenho de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Células HCT116 , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
18.
Elife ; 72018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30324906

RESUMO

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , Mycobacterium smegmatis/metabolismo , Peptidoglicano/metabolismo , Aminoácidos/metabolismo , Aminoaciltransferases/metabolismo , Amoxicilina/farmacologia , Bacillus/metabolismo , Parede Celular/metabolismo , Escherichia coli/metabolismo , Fluorescência , Cinética , Meropeném/farmacologia , Viabilidade Microbiana , Modelos Biológicos , Mycobacterium smegmatis/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/química
19.
PLoS Genet ; 14(10): e1007726, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30335755

RESUMO

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Actinas/metabolismo , Proteínas de Bactérias/genética , Ciclo Celular , Parede Celular/metabolismo , Citocinese/fisiologia , Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Morfogênese , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/metabolismo , Polimerização , Polissacarídeos/biossíntese
20.
J Biol Chem ; 293(46): 17985-17996, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30237166

RESUMO

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.


Assuntos
Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/metabolismo , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/biossíntese , Ácidos Teicoicos/metabolismo , Tioléster Hidrolases/metabolismo , Alanina/genética , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono-Oxigênio Ligases/metabolismo , Proteínas de Transporte/metabolismo , Ensaios Enzimáticos , Histidina/química , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Serina/química , Staphylococcus aureus/enzimologia , Tioléster Hidrolases/química , Tioléster Hidrolases/genética
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